Production of color in fish is generally limited to the dermal and epidermal regions of the skin, where chromatophores specialize in the synthesis and storage of light absorbing pigments (Fujii 1993; reviewed in Leclercq et al. 2010). In the case of walleye, Sander vitreus, color in some populations is a result of both the underlying pigmentation of the chromatophores and a blue color attributed to mucus on the outside of the fish (Regier et al. 1969, Scott and Crossman 1973). More recently, blue and yellow walleye morphotypes have been described from central Quebec, Canada (Paradis 2005) and northwestern Ontario, Canada (Yu et al. 2007) that also include blue mucus.
Native Sandercyanin Protein.
The inventors have isolated and described Sandercyanin, a novel blue protein derived from the mucus of walleye in the Papaonga River system of Ontario (Yu et al. 2007). The ecological significance of Sandercyanin has not been determined; however, it is secreted by the fish into its skin mucus. The Sandercyanin protein is a bili-binding, lipocalin protein with a molecular mass of 87,850. It is a tetramer with a subunit molecular mass of 21,386 Daltons. The Sandercyanin protein has absorption maxima at 280, 383 and 633 nm and has emission maxima at 678 nm on excitation at 380 nm and 630 nm (Yu et al. 2007). Both excitation and emission peaks are broad and have minimal spectral overlap.
Recombinant Proteins.
The importance of recombinant proteins for modern medical applications and therapy is known in the art. Recombinant production methods for bacteria are well developed and many important commercial proteins are produced in bacterial prokaryotic systems.
Recombinant DNA technology is one way of studying the functions and interactions of proteins. This is done by isolating a target DNA sequence and then transferring it to a cloning vector that has the ability to self-reproduce. The DNA of the cloning vector interacts with the target DNA and produces a new blueprint of gene information called recombinant DNA. The recombinant DNA is transcribed to RNA, which in turn produces the recombinant protein.
Color-Producing Compounds.
Bilin pigments, when associated with proteins, exhibit a wide variety of photophysical properties, i.e., intense fluorescence, photochemical interconversions, and radiation-less de-excitation. Differences in the protonation state, conformation and/or ionic environment of bilin pigments can significantly alter their absorption and emission properties. In this way, the protein moiety of bili-proteins tunes the spectrum of their bilin chromophore.
Plants, some bacteria, and fungi contain phytochromes, which are self-assembling bili-proteins that act as light sensors to modulate growth and development. Phytochromes' covalently bound bilin prosthetic groups photo-isomerize upon absorption of light, enabling the protein to photo-interconvert between two distinct species, which have absorption maxima in the red and NIR region. Unlike the intensely fluorescent phycobili-proteins, components of the photosynthetic antennae of algae, native phytochromes are non-fluorescent bili-proteins because this photo-conversion process is so efficient.
The optical properties of phytochromes are highly malleable, as shown by the spectral diversity of phytochromes in nature. In plants, algae and cyanobacteria, phytochromes are associated with the linear tetrapyrroles phytochromobilin (P.phi.B) or phycocyanobilin (PCB). Binding of an apo-phytochrome to the unnatural bilin precursor, phycoerythrobilin (PEB) however, affords a strongly fluorescent phytochrome known as a phytofluor, that is unable to isomerize upon light absorption (Murphy 1997). Phytofluors have been shown to be useful probes in living cells; however, addition of exogenous unnatural bilin precursors is generally necessary. Recently, a new class of phytochromes from bacteria and fungi was identified that attach a different bilin chromophore, biliverdin (BV), to an apparently distinct region of the apoprotein (Lamparter et al. 2002). These studies indicate that molecular evolution has occurred in nature to produce phytochrome mutants with novel spectroscopic properties.
Fluorescent Proteins.
Fluorescent proteins can be found in most molecular biology laboratories and have revolutionized the study of biology. Fluorescent probes are attractive due to their high sensitivity, good selectivity, fast response and their visual detectability. For example, the jellyfish green fluorescent protein (GFP) has revolutionized cell biological studies, allowing for the visualization of protein dynamics in real-time within living cells by in-frame fusion to a gene of interest. Other fluorescent proteins known to the art include Aequorea coerulescens GFP (AcGFP1), a monomeric Green Fluorescent Protein with spectral properties similar to those of EGFP (Enhanced Green Fluorescent Protein); tdTomato, an exceptionally bright and versatile red fluorescent protein that is 2.5 times brighter than EGFP; mStrawberry, a bright, monomeric red fluorescent protein which was developed by directed mutagenesis of mRFP; mRaspberry, developed by directed mutagenesis of mRFP1, a monomeric mutant of DsRed; E2-Crimson, a bright far-red fluorescent protein that was designed for in vivo applications involving sensitive cells such as primary cells and stem cells; DsRed-Monomer, an ideal fusion tag which has been expressed as a fusion with a large panel of diverse proteins with diverse functions and subcellular locations; and more.
Applications of fluorescent proteins include investigation of protein-protein interactions, spatial and temporal gene expression, assessing cell bio-distribution and mobility, studying protein activity and protein interactions in vivo, as well as cancer research, immunology and stem cell research and sub-cellular localization. Fluorescent proteins have also been used to label organelles, to image pH and calcium fluxes, and to test targeting peptides (Chiesa et al. 2001).
Despite their utility, as with any technology, existing fluorescent proteins have inherent limitations. For instance, GFP produces cytotoxic hydrogen peroxide (Cubitt et al. (1995)). Further, some fluorescent proteins are typically homo-dimers, a property that can interfere with the native function of the fused protein of interest. GFPs are also temperature and pH-sensitive and can be highly susceptible to photobleaching and oxidation. Further, GFPs are unable to fold and fluoresce in periplasmic/extra-cellular space (Jennifer et al. (2010)), hence finding limitation to be used for studying cell dynamics in the extracellular matrices.
Accordingly, a need exists for new methods of using fluorescent proteins.